Metal ions are ubiquitous in nature and are essential components of all biological systems. Cells utilize metal ions for a wide variety of functions, such as messengers, gas careers, catalysts, templates for polymer formation and regulatory elements for gene transcription.
Due to their importance and prevalence in industrial applications and manufacturing, metals have also become a major source of environmental contamination. The most toxic metal ions in environmental samples are those that are readily soluble in water. Unfortunately, current detection methods for soluble metal ions require bulky or sensitive instrumentation, making measurements in the field impractical. As a result, environmental samples must currently be transported to an analytical laboratory, resulting in a greater potential for contamination or sample degradation during storage. It is known that storage can alter the metal-ion concentration of a sample through adsorption of the metal on the surface of the container, or by the growth of microorganisms that can alter the speciation of metal ions in the sample. Fluorescent reagents that can simply and cheaply detect a variety of metal ions, including the ability to differentiate different oxidation states of the same metal ion, in a variety of solutions (biological, environmental, industrial), would provide an economical and reliable means for the detection of such environmental contaminants in the field.
Metal chelators that contain heteroaromatic nitrogen atoms are well known in the scientific literature, and the stability constants and properties of these chelators have been thoroughly reviewed and compiled (e.g. Martel et al., CRITICAL STABILITY CONSTANTS, VOL. 1, Plenum Press, New York (1974)). Molecules with heterocyclic nitrogen binding sites are widely used as analytical tools for extracting metal ions into organic solvents. Chemists currently take advantage of the extreme water insolubility of the majority of these chelators to preconcentrate metal ions in organic solvents prior to atomic absorption spectroscopy or colorimetric detection. However, these chelators are generally not water soluble and could not be used in biological systems. Further, they do not function as fluorescent indicators for the metal ions they complex.
Fluorescent indicators for metal ions that comprise a nitrogen-containing heterocycle as the ion binding site and an additional covalently attached fluorophore as a responding element have not been described in the literature. While a variety of fluorescent indicators that are useful for the detection of biologically relevant soluble free metal ions (such as Ca.sup.2+, Mg.sup.2+ and Zn.sup.2+) have been described, these indicators utilize oxygen-containing anionic or polyanionic chelators to bind to metal ions. In particular, fluorescent indicators utilizing a polycarboxylate BAPTA chelator have been previously described (U.S. Pat. No.: 4,603,209 to Tsien et al., 1986; U.S. Pat. No. 5,049,673 to Tsien et al., 1991; U.S. Pat. No. 4,849,362 to DeMarinis, et al. 1989; application REACTIVE DERIVATIVES OF BAPTA USED TO MAKE ION-SELECTIVE CHELATORS, Ser. No. 07/843,360, filed Feb. 25, 1992, allowed Sep. 21, 1994 as U.S. Pat. No. 5,453,517; application BENZAZOLYLCOUMARIN-BASED ION INDICATORS, Ser. No. 08/247,013, filed May 20, 1994, now U.S. Pat. No. 5,501,980; application BENZAZOLYLCOUMARIN-BASED ION INDICATORS FOR HEAVY METALS, Ser. No. 08/246,847, filed May 20, 1994, now U.S. Pat. No. 5,459,276). While some of these known indicators have been used to detect divalent or trivalent ions other than Ca.sup.2+ or Mg.sup.2+, previously known polycarboxylate fluorescent metal ion chelators usually suffer from high sensitivity to micromolar concentrations of Ca.sup.2+ ion, or millimolar concentrations of Na.sup.+ or K.sup.+. This is an extreme drawback when it is desirable to detect extremely small concentrations of metal ions in the presence of other metal ions, such as in biological fluids or sea water, or any sample that contains Ca.sup.2+, Na.sup.+ or K.sup.+. These fluorescent indicators are consequently unable to discriminate toxic metal contamination in the presence of these common ions. Several fluorescent indicators selective for Li.sup.+, Na.sup.+ and K.sup.+ in aqueous or organic solution have been described, but their metal binding and ability to discriminate ions has usually been based on the chemical modification of crown ethers (U.S. Pat. No. 5,134,232 to Tsien et al., 1992; UK patent publication GB 2,277,096 A, to Kuhn et al., published Oct. 19, 1994).
A wide variety of other metal complexing agents that either yield fluorescent metal complexes or have their intrinsic fluorescence quenched by complexing with a metal have been described. These have been extensively reviewed by Guilbault in PRACTICAL FLUORESCENCE, 2nd Edition, Marcel Dekker, Publishers (1990). Many of these reagents require extraction of the metal into an organic solvent prior to its detection or quantification. Also, metal detection by many of the reagents involves a reduction in fluorescence of the reagent rather than the experimentally preferred enhancement of fluorescence. Metals have been determined by the selective extraction of the ternary complex of a metal, a fluorophore and a complexing agent into an organic solvent. This method differs from the subject invention both in requiring a three component mixture of reagents and in requiring extraction into an organic solvent. Metal complexes of certain reagents, including phenanthridines and bipyridyls, are intrinsically luminescent, particularly in organic solvents, as a result of emission transitions of the metal itself and the complexing agent primarily serves to protect the metal from quenching by solvent and to absorb the exciting light. These are typically complexes of ruthenium or certain rare earth metals. Various methods recommended for detection of metals by the catalytic formation of a fluorescent product differ from the instant method in that such catalytic methods lack a rapid and reversible response.
In general, a useful property for metal ion indicators is the ability to detect and/or quantify a selected metal ion in the presence of other metal ions. While discrimination from Ca.sup.2+, Na.sup.+ and K.sup.+ ions (as discussed above) is useful for certain biological or environmental samples, the ability to discriminate other metal ions is also useful. In particular, an indicator that could differentiate between different oxidation states of a single metal would be very useful for detecting oxidative or reductive activity. For example, the detection of Fe.sup.2+ in the presence of Fe.sup.3+, or the detection of Cu.sup.+ in the presence of Cu.sup.2+.
The indicators of the present invention allow the direct measurement of trace concentrations of selected metal ions in solution, including the measurement of specific oxidation states. The indicators described are insensitive to high concentrations of monovalent and divalent ions commonly found in seawater and biological fluids, and therefore can be used to assay metal ions such as Hg.sup.2+, Pb.sup.2+ or Cu.sup.2+ in the range of 10.sup.-6 to 10.sup.-9 molar, or lower, even in the presence of high concentrations of Ca.sup.2+, Mg.sup.2+, K.sup.+, Na.sup.+ and Cl.sup.-. The sensitivity and discrimination of the instant indicators lets solution assays be performed without requiring preconcentration or other manipulations of the sample. The need to manipulate the sample in this way when using other, less sensitive detection reagents, is one of the main sources of error in determining metal contamination of environmental samples. Finally, several of the indicators of the present invention allow the researcher to detect metal ions using only ambient light or low cost, battery powered UV excitation sources, ideally suited for field conditions. The indicators are therefore highly useful for on-site environmental testing, eliminating the difficulties associated with sampling, sample transportation and sample storage.
In general, the fluorescent indicators of the present invention serve as direct, sensitive probes for heavy metal ions at the very low (parts-per-million (ppm), parts-per-billion (ppb)) concentrations generally associated with deleterious biological effects and environmental regulation.